Core Director: Yvette Taché, Ph.D., Professor, Division of Digestive Diseases, Department of Medicine, UCLA

Core Co-Director: Jonathan Kaunitz, M.D., Professor, Division of Digestive Diseases, Department of Medicine, UCLA

Core Associate Director: Million Mulugeta, D.V.M., Ph.D., Associate Researcher, Division of Digestive Diseases, Department of Medicine, UCLA

OBJECTIVES

The overall objectives of the Animal Models Core are to provide facilities and expertise to researchers for in vivo characterization of normal and pathophysiological mechanisms of hormonal and neural regulation of GI function and brain-gut interactions. The specific objectives are to provide CURE investigators with: (1) in vivo experimental models to assess gastric, pancreatic, and intestinal function in rats and mice; (2) specialized facilities and equipment for measuring gastrointestinal secretory and motor functions, and mapping brain neuronal activity and extrinsic nerve activity; (3) methods for in vivo administration of peptides, neurotransmitters, and drugs, sampling blood and body fluids, and removing tissues; (4) methods to assess afferent and efferent arms of the pathways in brain-gut interactions; and (5) expertise in protocol design and data interpretation for in vivo studies.

CORE SERVICES

A. GASTRIC ACID SECRETION, BLOOD FLOW, AND MUCOSAL BARRIER FUNCTION

A 1. Urethane-anesthetized rats
Acid output is monitored at set time intervals (2 10 min) through an acutely implanted catheter by continuous intragastric perfusion and back-titration or by collection of gastric contents. This model has the advantage of providing consistently low basal secretory rate and reliable assessment of responses to exogenous stimulants of gastric acid secretion given either peripherally (intravenously, intra-arterially, intraperitoneally) or centrally (intracerebroventricularly, intracisternally or microinjection into the brain parenchyma), and to endogenous stimulants, such as an intragastric meal. In addition, inhibitors of stimulated gastric acid secretion can be assessed. Examples of uses for this model include bioassay of newly characterized peptides or molecular forms of peptides; determinations of the mechanism or pathway of action of endogenous peptides, such as in intestinal feedback inhibition of gastric acid secretion; and studies to determine the locus of action, such as central versus peripheral sites. In addition, measurements of gastric acid secretion can be combined with monitoring of other gastrointestinal parameters (gastric mucosal blood flow, gastric motor function) (35).

A 2. Urethane-anesthetized mice
This is essentially the same model as described above but modified for mice and recently developed by the Core. Acid output is monitored at set time intervals (2 10 min) through an acutely implanted catheter by continuous intragastric perfusion and back-titration. The anesthetized mouse model provides a method for study of gastric secretory physiology, which previously has only been examined in this species using an ex vivo stomach preparation. In contrast to such vascularly and luminally perfused stomach preparations, this in vivo model maintains intact all hormonal and intrinsic and extrinsic neural connections to the stomach and, most importantly, can be used in transgenic mice in which genes for peptides and receptors important in the regulation of gastric function have been altered. Using somatostatin receptor 2 knockout mice, we showed the key role of this signaling pathway in urethane- and bombesin-induced inhibition of gastric acid secretion (17,28).

A 3. Awake(unanesthetized) rats
Gastric acid secretion is measured by gravity drainage or by continuous intragastric perfusion and back titration via a chronic gastric cannula in awake rats accustomed to light restraint in Bollman cages. This model has the obvious advantage of measuring function in awake animals allowing us to assess the cephalic phase of gastric function and of being cost effective in that these rats can be used repeatedly over several months (14).

B. GASTRIC AND INTESTINAL MOTOR FUNCTION

B 1. Gastric emptying
Emptying of a non nutrient liquid solution in awake rats and mice. Gastric emptying is measured at different time points (20-60 min) after administration of a semi-viscous liquid (methylcellulose) containing a non absorbable dye (phenol red) marker. Gastric emptying is calculated from the amount of phenol red remaining in the stomach, taking into consideration the amount initially administered. This method is useful for defining the effects of short acting exogenous peptides or other gastrointestinal stimuli, administered either peripherally or centrally (13,23).

Emptying of solid chow after a short fast in awake rats and mice. Animals are deprived of food for 18-24 h, then have free access to preweighed solid Purina Chow for a set period of time (1 to 3 h). The food is then removed, and gastric emptying can be determined over several hours by measuring the wet weight of the stomach (15,33). As gastric emptying of liquid non nutrient and solid nutrient meals are regulated differently, this is a complementary approach to assess the role of nutrients in the regulation of gastric motor function.

B 2. Gastrointestinal motor function


Fig. 1. Pattern of response to i.v. injection of CCK-8 (3 μg/kg) in urethane anesthetized rats. The intragastric pressure (IGP) dropped, as pylorus (PYL), circular muscles in the corpus (CORci), longitudinal muscles in the corpus (CORln) contracted and lower esophageal sphincter (LES) separated. Prevailing rhythmic activity was inhibited during the tonic response, and increased in both frequency and amplitude upon recovery. Note that LES closed well before PYL re-opened to pre-injection levels (from ref. 2 in press).

Ultrasonomicrometry recording in rats and mice. Methods commonly used to monitor motility in the rat - EMG recordings, strain gauge recordings, or manometric techniques - are much more difficult in the mouse, and are not at present in common usage. This is largely a result of the delicacy and small size of mouse tissues. The ultrasonometric measurement to detect motion changes in the gut muscles has recently been developed (2). The ability to examine simultaneously motility changes in specific GI structures and sphincters has recently permitted us to discriminate a consistently more rapid re-closure of lower esophageal sphincter (LES) than re-opening of pyloric sphincter (PYL) following intravenous injection of CCK-8 (Fig. 1) (2). It also allowed us to discriminate between the activity of circular and longitudinal muscles (Fig. 1).
The simultaneity of recording within the various components of the GI tract obtained by ultrasonomicrometric measurements can provide valuable information on the coordinated motion of the GI tract. Mice or rats are anesthetized using urethane (1.5 g/kg, i.p.), abdominally shaved, and placed on a feedback-controlled heating plate to maintain rectal temperature in the range 36.5-37.5oC. The trachea and jugular vein are cannulated Animals are kept hydrated via continuous i.v. infusion of sterile saline through the jugular vein, as previously reported (22). After laporotomy, ultrasonometric piezoelectric crystals (Sonometrics Corp., London, OT) are affixed as pairs 2-6 mm apart using cyanoacrylate glue to the serosal surface of structures of interest; these include the LES, the antrum, the pylorus, the cecum, the proximal colon, and the distal colon. In antrum, cecum, proximal and distal colon, three crystals will be placed, orienting them such that one pair of crystals is oriented parallel to the circular muscle, and another is oriented parallel to the longitudinal muscle. Due to the much smaller size of the mouse relative to the rat, only two pairs will be used in each animal, and the size of the crystal is reduced to 0.7 mm in mice versus 1 mm in rats. The abdominal incision is closed, and the animals are allowed to stabilize for at least forty-five minutes.
Sonometric distance measurement data will be acquired digitally at 50 samples/s using a digital sonomicrometer (TRX-13, Sonometrics Corp, London ONT) connected to a Pentium III class computer running SonoLAB software (Sonometrics Corp, London, ONT). Digitally acquired distance data are simultaneously output as analog signals via an installed 4-channel DAC. These sonometric analog signals, along with analog physiological data (rectal temperature, EKG), are acquired using a Micro1401 A/D interface (Cambridge Electronic Design, Ltd, Cambridge) connected to a Pentium II class computer running Spike 2 (Cambridge Electronic Design, Ltd, Cambridge) data acquisition software. Data obtained will be analyzed using software written in our laboratory using the Spike 2 scripting system. Activity in each region is characterized in terms of frequency of phasic (i.e. ongoing rhythmic) activity, amplitude of phasic activity, and fluctuations in both phasic activity and underlying tone (2).

B 3. Colonic motor function

Distal colonic transit in awake rats and mice. Under short isoflurane anesthesia a small bead is inserted into the rat or mice distal colon as previously reported (22,25) and the time to expel the bead is monitored (22,25).

C. MUCOSAL DEFENSE MECHANISMS

C 1. Mucosal Blood Flow
The laser-Doppler method with either straight or right-angle probe is placed on the mucosa or on the serosa of the gastric or duodenal epithelium of anesthetized rats or mice in order to measure relative mucosal blood flow (10).

C-2. Mucus Gel thickness and secretion
Mucus gel thickness can be measured over the gastric or duodenal epithelium of anesthetized rats or mice by measuring the distance between focal planes of the mucosal surface and of the surface of the mucus gel, as delineated by carbon particles or fluorescent microbeads. Mucus secretion is measured in the effluent of perfused duodenum by measuring the reaction to alcian blue (30).

C 3. Intracellular pH
Intracellular pH of gastric or duodenal epithelial cells or anesthetized rats or mice can be measured by isolating the exposed gastric or duodenal mucosa in a perfusion chamber, loading the mucosa with a pH-sensitive fluorescent dye (BCECF-AM), and imaging the mucosa with fluorescent life animal ratio imaging (FLARM) using an epifluorescent microscope, filter wheel, and image analyzer (29).

Fig. 2 Fluorescent confocal (A-C) and conventional (D) images of a pH-sensitive dye loaded into the duodenal epithelial cells of urethane-anesthetized mice. This technique enable the core to measure intracellular ion concentrations or any fluorescent marker in live mice. (submitted to Gastroenterology).

D. MEASUREMENT OF FOOD INTAKE

D-1. Freely feeding solid food intake in rats and mice Animals are trained to eat daily during a set time period (0900-1200 h) and are studied during this time period. For the remaining 21 h, animals are deprived of food but not water. Fasted rats or mice are given free access to preweighed chow. Food intake is determined by measuring the difference between the preweighed and the remaining weight of chow and the spill. Measurements can be taken every hour or at the end of a predetermined time period (4,33).

D-2. Sham-feeding in rats
Rats are fitted with a chronic gastric cannula and accustomed to light restraint in Bollman cages and to feeding in the restraint cage. For experiments, rats are fasted and exposed to the smell and the sight of chow pellet without access. We showed that 30 min exposure to sham feeding stimulates gastric acid secretion with a peak response at 20 min as monitored by automatic continuous titration of the intragastric perfusate (14).

E. ELECTROPHYSIOLOGICAL RECORDING OF VISCERAL NERVE ACTIVITY

Electrical activity from vagal afferent or efferent single fibers or multi unit activity in the different subdiaphragmatic branches of the vagus or splanchnic afferent fibers is performed in urethane anesthetized rats. The response of units to mechanical (distension, stretch) or chemical (duodenal or gastric perfusion) stimuli or to intravascular administration of drugs and peptides can be assessed. The number of spikes per unit time is measured by a rate-meter and single units can be discriminated on the basis of spike height using dedicated software. This experimental approach is useful to monitor changes in vagal afferent and efferent activity in response to central or peripheral stimuli or transmitters as well as a variety of physiological, pathophysiological and pharmacological conditions (3,12).
A novel dual simultaneous measurement of gastric vagal afferent and efferent activity has also been developed by Dr. Adelson (1)..

F. FUNCTIONAL MAPPING OF NEURONAL ACTIVATION

In a variety of models, the c-fos proto-oncogene mRNA and its product, the c- Fos protein, have been shown to be expressed in response to neuronal activation. This observation can be exploited to trace neuronal activation by visceral noxious, physiological stimulation and various stressors. After the appropriate experimental procedure (for example, administration of a peptide or nutrient or surgical intervention), rats or mice are euthanized at various time points up to two hours and transcardially perfused. Neural tissue is removed and processed for Fos-like immunoreactivity using immunocytochemistry with a commercially available antibody for Fos protein. The Animal Core has been the first to adapt this approach for functional mapping of brain-gut interactions (5), and the method has been used extensively in rats and mice since then (21,32,34).

G. EXPERIMENTAL ANIMAL MODELS

In addition to the techniques described above, more complex experimental models are available to CURE: DDRCC members through the Animal Models Core. Many of these have been developed by the Core and are useful for the study of perturbation in the physiology of the gastrointestinal system.

G 1. Gastroduodenal injury and mucosal defense models The following experimental mucosal lesion models in rats are available to CURE: DDRCC investigators. Both acute and chronic models of gastric mucosal lesions are available, including ethanol, intracisternal injection of TRH, and chronic ulcers induced by acetic acid (8,9).

G-2. Visceral pain in rats and mice
Behavioral responses to graded distension of the colon or rectum can be measured in the form of abdominal contractions or measurement of abdominal wall EMG in fasted, awake rats. Hyperalgesia can be induced by a regimen of conditioning stimulus (repeated colorectal distension) in rats and mice (20,21,26).

G-3. Experimental colitis in rats and mice Trinitrobenzen-sulfonic acid (TNB) is dissolved in 50% ethanol at a concentration of 120 mg/ml. A total volume of 0.25 ml of the solution is injected into the rectum of anaesthetized rats. Rats are placed in restraint cages and tipped head down to an angle of 45% to minimize leakage of TNB from the rectum. Colitis is evaluated after 7 days by histology, macroscopic examination, and myeloperoxidase activity (11,27).
G-4. Surgically-induced ileus in rats and mice This can be performed in both rats and mice. Animals are anesthetized with halothane, and a midline abdominal incision performed. The cecum is removed, covered by a warm moistened cotton swab and then lightly manipulated or tapped for 1-5 min. The cecum is replaced in the abdominal cavity and the peritoneum and skin incision closed. Colonic transit, whole gut transit or gastric emptying can then be measured using the techniques described above (13,18).

G-5. Stress induced alterations in gastrointestinal function

A model of psychological stress is available that is not ulcerogenic but induces profound alterations of gastrointestinal motor function (stimulation of colonic transit and fecal output) and ACTH release. Rats are placed on a small platform surrounded with water for one hour or restrained for one to three hours at room temperature. The strengths of this model are its reliability in stimulating colonic motor function, that it does not require sophisticated equipment, and that it is not painful. This model developed and characterized by the animal Core (6,19) is now largely used by CURE: DDRCC investigators (Dr. Mayer) and other investigators (Drs. Perdue, Malagelada). Other models are restraint stress in rats and mice (25).

G-6. Cephalic, gastric, and intestinal phases of gastric acid secretion in rats

Cephalic phasic acid secretion. This can be achieved by injection of thyrotropin releasing factor (TRH) into the cisterna magna in rats under brief isoflurane anesthesia or the dorsal motor nucleus of the vagus in urethane anesthetized rats (14). In conscious rats, TRH is injected through chronically implanted cannulas into the lateral ventricle (16).

Gastric and intestinal phases of gastric acid secretion. A model has been developed to study the gastric phase of acid secretion in rats using peptone solution placed into the stomach through gastric cannulas in urethane and thiopental anesthetized rats and in conscious rats. For conscious rats, cannulas are placed into the forestomach and any point in the intestine (duodenum, jejunum, or colon) according to the needs of the experiment. Rats can be used repetitively for a period of three to four months after surgery. Surgically prepared, awake rats are trained to accept light restraint in Bollman cages for measurement of gastric parameters (gastric acid output and gastric motor function) (31).
Direct measurement of acid permeation into rat esophagus. In order to better understand the dynamics of acid permeation into the esophageal mucosa, a new method has been developed to measure interstitial pH (pH(int) of the esophageal basal epithelial layer, pre-epithelial layer thickness, and blood flow in rats in vivo during luminal acid challenge. A novel confocal microscopic technique is used in vitro to measure pH(int) from defined cellular sites in response to luminal and basolateral acidification. Using 5-(and-6)-carboxyfluorescein (CF) and carboxy-seminapthorhodofluor-1 (SNARF-1) fluorescence, the pH(int) is measure by conventional and confocal microscopy in urethane anaesthetized rats. Pre-epithelial layer thickness was measured optically with carbon particles as markers. Blood flow was measured with laser Doppler flowmetry (29).

G-7. Transgenic mice
CURE: DDRCC members have access to a colony of CFTR mutant mice maintained at UCLA that serve as interesting models of duodenal barrier function. Other planned transgenic mice include those with deletions of genes involved with duodenal bicarbonate secretion and pH homeostasis such as NHE3. We also have access to transgenic mice by liaison with Dr. C. Evans, Director of the UCLA Opioid Receptors and Drug Abuse Mutant Animal Breeding Core (selective opiate receptor knockout mice), Salk institute (CRF receptor knockout mice) (13) as well as Dr. Meisheng Jiang, who was CURE:DDRCC Named New Investigator and has been newly appointed in the Department of Molecular and Medical Pharmacology, David Geffen School of Medicine at UCLA and nominated Director of the UCLA Transgenic Mouse Facility. This Transgenic Facility produces transgenic and knockout mice in a specific pathogen free (SPF) environment and offers additional related services for investigators at UCLA. The Transgenic Mouse Facility is equipped for the collection, microinjection, and re-implantation of fertilized mouse eggs and blastocysts.

H. OTHER EXPERTISE FOR ANIMAL HANDLING OR SURGICAL PROCEDURES

H 1. Drug delivery
Drugs can be administered into the stomach, blood (portal, arterial, venous), cerebrospinal fluid, specific brain nuclei, peritoneal cavity, subcutaneously, intrathecally and perineurally (cervical vagal, celiac ganglia) according to established and routine methodology.

H-2. Teaching or performing specialized assay methods or use of specialized equipment Core personnel are available to teach or perform standard and specialized assay procedures to CURE: DDRCC members. Examples include acid or duodenal bicarbonate secretion, myeloperoxidase, longitudinal myenteric preparation (7,24,27).

H 3. Collection of body fluid/tissues
Body fluids can be collected from anesthetized rats and mice from the arterial or venous systems, portal vein, cerebrospinal fluid, and saliva. Many different tissues can be harvested from deeply anesthetized animals for peptide extraction or other uses, including but not limited to tissue from the gastrointestinal tract. More specialized procedures involve removal of neural tissue for primary cell culture.
H 4. Surgical preparations
Chronic cannulas can be implanted into specific brain nuclei, lateral brain ventricles, stomach, duodenum, and proximal colon in rats. Surgical denervations and capsaicin treatments can be performed.

H-5. Expertise in study design, data analysis and interpretation Core personnel are available to CURE: DDRCC members to advise on all aspects of conducting research that uses animals and experimental animal models.